The three-dimensional organization of ribosomal genes and the architecture of the nucleoli vary with G1, S and G2 phases.

The three-dimensional (3-D) organization of the nucleolus, a defined nuclear territory containing tandem repeats of the ribosomal genes (rDNA), was investigated in PtK1 cells. Identification of the interphase stages was performed in single cells using DNA quantification by cytometry for the G1 and G2 phases while the S phase was identified by immunolabelling of the proliferating cell nuclear antigen (PCNA). The 3-D organization of the rDNA in the nucleolus was analyzed by fluorescence in situ hybridization using confocal microscopy. All the rDNA was located inside the nucleolar structures during all stages and the two rDNA loci were orthogonal. The rDNA was heterogeneously distributed in each nucleolus during G1, S and G2, with alternate sites of clustered genes (spots) and of genes in more extended configurations. The number of spots (4 to 6 in G1) increased during S phase (7 to 12) and their 3-D organization was progressively relaxed from G1 to G2. Double spots in G2 could reflect a similar gene organization of two chromatids. During mid-S phase, PCNA co-localized with some clustered genes (spots), indicating that rDNA replication occurs inside nucleoli and at different sites of the same locus simultaneously. The evaluation of the rDNA transcription units in 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB)-treated cells indicated a mean of 16 units per G1 nucleus and 25 units per G2 nucleus. For G1 and G2, the fine 3-D structure of nucleoli was reconstructed using ultrathin serial sections after specific contrast of DNA and RNA, digitization of the serial section images and computer-assisted 3-D architecture. Fibrillar centers (FCs) formed discrete structures (about 10 in G1 and 20 in G2) connected by a network of the dense fibrillar component. The 3-D arrangement of the FCs in G1 and G2 are similar to the rDNA spots. In conclusion, the architecture of the nucleoli during interphase reflects the distribution of the rDNA that is characterized by alternation of clustered and extended genes.

[1]  F. Wachtler,et al.  The functional significance of nucleolar structures. , 1991, Annales de genetique.

[2]  P. Cook,et al.  Site of transcription of ribosomal RNA and intranucleolar structure in HeLa cells. , 1994, Journal of cell science.

[3]  C. Got,et al.  Molecular dissection of a specific nuclear domain: the chromatin region of the ribosomal gene cluster in Xenopus laevis. , 1992, Experimental cell research.

[4]  F S Fay,et al.  A three-dimensional view of precursor messenger RNA metabolism within the mammalian nucleus. , 1993, Science.

[5]  M. Marilley,et al.  Supercoiled loop organization of genomic DNA: a close relationship between loop domains, expression units, and replicon organization in rDNA from Xenopus laevis. , 1989, Experimental cell research.

[6]  D. Jackson,et al.  Replication and transcription sites are colocalized in human cells. , 1994, Journal of cell science.

[7]  S. Ghosh,et al.  The nucleolar structure. , 1976, International review of cytology.

[8]  D. Hernandez-Verdun,et al.  Localization of the RNA polymerase I transcription factor hUBF during the cell cycle. , 1993, Journal of cell science.

[9]  A Soyer,et al.  Computer reconstruction of nucleolar architecture by interactive three-dimensional color display. , 1991, Journal of electron microscopy technique.

[10]  R. Ochs,et al.  Electron microscopic localization of ribosomal DNA in rat liver nucleoli by nonisotopic in situ hybridization. , 1993, Experimental cell research.

[11]  Peter Shaw,et al.  Different patterns of rDNA distribution in Pisum sativum nucleoli correlate with different levels of nucleolar activity , 1993 .

[12]  G J Brakenhoff,et al.  Dynamics of three-dimensional replication patterns during the S-phase, analysed by double labelling of DNA and confocal microscopy. , 1992, Journal of cell science.

[13]  M. Garcia-Blanco,et al.  Nuclear spreads: I. Visualization of bipartite ribosomal RNA domains , 1995, The Journal of cell biology.

[14]  J. Dumont,et al.  Demonstration of cell cycle kinetics in thyroid primary culture by immunostaining of proliferating cell nuclear antigen: differences in cyclic AMP-dependent and -independent mitogenic stimulations. , 1993, Journal of cell science.

[15]  A. Houtsmuller,et al.  The spatial localization of 18 S rRNA genes, in relation to the descent of the cells, in the root cortex of Petunia hybrida. , 1994, Journal of cell science.

[16]  U. Scheer,et al.  Localization of RNA polymerase I in interphase cells and mitotic chromosomes by light and electron microscopic immunocytochemistry. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[17]  E. Jordan,et al.  Widely dispersed DNA within plant and animal nucleoli visualised by 3-D fluorescence microscopy , 1992, Chromosoma.

[18]  D. Rawlins,et al.  Localization of ribosomal and telomeric DNA sequences in intact plant nuclei by in‐situ hybridization and three‐dimensional optical microscopy , 1990, Journal of microscopy.

[19]  J. Sylvester,et al.  A study on nucleolar DNA: isolation of DNA from fibrillar components and ultrastructural localization of different DNA probes. , 1993, Journal of cell science.

[20]  K. Smetana,et al.  Three‐dimensional reconstructions of nucleolus‐organizing regions in PHA‐stimulated human lymphocytes , 1989, Biology of the cell.

[21]  J. S. Heslop-Harrison,et al.  Different patterns of rDNA organization at interphase in nuclei of wheat and rye. , 1992, Journal of cell science.

[22]  M. Sánchez-Pina,et al.  A specific ultrastructural method to reveal DNA: the NAMA-Ur. , 1991, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[23]  Y. Usson,et al.  Intranuclear co-location of newly replicated DNA and PCNA by simultaneous immunofluorescent labelling and confocal microscopy in MCF-7 cells. , 1992, Journal of cell science.

[24]  M. V. Semenov,et al.  The RNA polymerase I-specific transcription initiation factor UBF is associated with transcriptionally active and inactive ribosomal genes , 1993, Chromosoma.

[25]  U. Scheer,et al.  A possible mechanism for the inhibition of ribosomal RNA gene transcription during mitosis , 1995, The Journal of cell biology.

[26]  P. Hozák,et al.  Quantitative ultrastructural study of nucleolus‐organizing regions at some stages of the cell cycle (G0 period, G2 period, mitosis) , 1988, Biology of the cell.

[27]  A. Solari,et al.  Methanol-Acetic anhydride: an efficient blocking agent for electron microscope cytochemistry. Its application to mouse testis and other tissues , 2004, Histochemistry.

[28]  R. Loppes,et al.  Three-dimensional electron microscopy of ribosomal chromatin in two higher plants: a cytochemical, immunocytochemical, and in situ hybridization approach. , 1991, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[29]  D. Hernandez-Verdun.  The nucleolus today. , 1991, Journal of cell science.

[30]  G. Goessens,et al.  Quantitative analysis of Ehrlich tumour cell nucleoli during interphase. , 1982, Experimental cell research.

[31]  M. Thiry,et al.  Localization of nucleolar chromatin by immunocytochemistry and in situ hybridization at the electron microscopic level. , 1991, Electron microscopy reviews.

[32]  P. Hozák.  Catching RNA polymerase I in Flagranti: ribosomal genes are transcribed in the dense fibrillar component of the nucleolus. , 1995, Experimental cell research.

[33]  J W Gray,et al.  Cytogenetic analysis using quantitative, high-sensitivity, fluorescence hybridization. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[34]  M. Méchali,et al.  Chromosomal replication initiates and terminates at random sequences but at regular intervals in the ribosomal DNA of Xenopus early embryos. , 1993, The EMBO journal.

[35]  G. Géraud,et al.  Three-dimensional organization of the ribosomal genes and Ag-NOR proteins during interphase and mitosis in PtK1 cells studied by confocal microscopy , 1993, Chromosoma.

[36]  M. Schmid,et al.  Quantitative determination of rDNA transcription units in vertebrate cells. , 1991, Experimental cell research.

[37]  P. Shaw,et al.  The nucleolar architecture of polymerase I transcription and processing. , 1995, The EMBO journal.

[38]  Prof. Dr. Asen A. Hadjiolov.  The Nucleolus and Ribosome Biogenesis , 1985, Cell Biology Monographs.

[39]  D. Ward,et al.  Delineation of DNA replication time zones by fluorescence in situ hybridization. , 1992, The EMBO journal.

[40]  B. Schlegelberger,et al.  Simultaneous fluorescence immunophenotyping and interphase cytogenetics: a contribution to the characterization of tumor cells. , 1992, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[41]  J. Lawrence,et al.  Probing functional organization within the nucleus: is genome structure integrated with RNA metabolism? , 1993, Cold Spring Harbor symposia on quantitative biology.

[42]  J. Celis,et al.  Cell cycle-dependent variations in the distribution of the nuclear protein cyclin proliferating cell nuclear antigen in cultured cells: subdivision of S phase. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[43]  G Geraud,et al.  Three‐dimensional computer reconstructions from serial sections of cell nuclei , 1988, Biology of the cell.

[44]  G. Brugal,et al.  Combination of DNA in situ hybridization and immunocytochemical detection of nucleolar proteins: a contribution to the functional mapping of the human genome by fluorescence microscopy. , 1994, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[45]  J. Bachellerie,et al.  Nucleolar organization of HeLa cells as studied by in situ hybridization , 1991, Chromosoma.